DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims Status
Claims 1-4 & 7-17 filed on 10/24/2025 are pending. The cancellation of claims 5 & 6 in the reply filed on 10/24/2025 is acknowledged. All the amendments and arguments have been thoroughly reviewed but are deemed insufficient to place this application in condition for allowance. The following rejections are either newly applied, as necessitated by amendment, or are reiterated. They constitute the complete set being presently applied to the instant application. Response to Applicant’s argument follow. This action is FINAL.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
Any rejection not reiterated is hereby withdrawn in view of the amendments to the claims.
Information Disclosure Statement
Only the abstract of the reference in the IDS submitted on 10/27/2025 that is lined through, under the non-patent literature section, was considered because an English copy of the full document was not provided.
Claim Rejections - 35 USC § 103
Claim(s) 1-4, 11, 16, & 17 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang et al.; ACS Nano, Vol. 9, pages 11812-11819, November 2015), in view of Crawford (WO 2015/150786 A1), as cited in the IDS dated 07/19/2022.
Regarding amended claim 1, Zhang teaches a method of preparing cross-linked DNA substrates through a reaction of the guanine residue with the aldehyde group of an abasic site on the opposing strand of the duplex DNA in the presence of NaCNBH3 (forming an inter-strand cross link between the first and second strands of the target double stranded nucleic acid by exposing the target double stranded nucleic acid to a cross-linking agent) and then examining these interstrand cross-links using a nanopore for the detection and characterization (sequencing the cross-linked construct using a single molecule sequencing technique) of the interstrand cross-links in duplex DNA (abstract lines 1-6; pg. 11813 paragraph bridging column 1 & 2 lines 1-7; pg. 11817 paragraph bridging column 1 & 2 lines 1-19; Table 1; Figure 2; Figure 4). Zhang also teaches that when higher voltage is applied the D3 cross-link (cross-linked construct) conformational adjusts to enable the cross-link to squeeze through the pore in which the double-stranded nucleic acid breaks in the vicinity of the interstrand cross-link, as seen in Figure 4d, and then allows the cross-linked duplex D3 (cross-linked construct) to translocate from the cis to the trans side of the nanopore (sequentially processing the first strand and the second strand of the cross-linked construct) (pg. 11814 paragraph bridging column 1 & 2 lines 2-4 & 24-36; Table 1; Figure 4).
Zhang does not teach that the cross-linked construct is contacted with an enzyme to sequentially process the cross-linked construct.
Crawford teaches a method of characterizing (sequencing) a target double stranded polynucleotide that has a hairpin adaptor on one end of the target double stranded polynucleotide to bind the first strand and second strand of the target polynucleotide by contacting the target polynucleotide with a transmembrane pore (nanopore) and a polynucleotide binding protein that is a polynucleotide handling enzyme capable of controlling the movement of at least one strand of the polynucleotide through the pore (contacting target double stranded polynucleotide with an enzyme) (abstract lines 1-3; pg. 1 lines 14-15; pg. 2 lines 18-21 & 26-31; pg. 41 lines 25-34; pg. 42 lines 1-17). Crawford also teaches that the polynucleotide handling enzyme may modify the target polynucleotide through orienting it or moving it to a specific position allowing the enzyme to control the movement of the target polynucleotide (pg. 41 lines 25-34; pg. 42 lines 1-17).
Zhang and Crawford are considered to be analogous to the claimed invention because they are all in the same field of sequencing target double stranded nucleic acids through a nanopore. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of sequentially processing the first strand and the second strand in the cross-linked D3 construct in Zhang to incorporate the contacting the target double stranded nucleic acid with an enzyme as taught in Crawford to provide orthogonal proof-reading sequence information because Crawford teaches that doing so would provide a means to control the movement of the target double stranded nucleic acid through the nanopore.
Regarding claim 2, Zhang teaches the that duplex D3 (cross-linked construct) is cross-linked only 4 bp from the end of the duplex and that when higher voltage is applied the D3 cross-link conformational adjusts to enable the cross-link to squeeze through the pore in which the double-stranded nucleic acid breaks in the vicinity of the interstrand cross-link as seen in Figure 4d (allowing the double stranded nucleic acid to break in the vicinity of the interstrand cross-link) which then places the interstrand cross-link in the vicinity of the terminus of D3 (vicinity of the terminus of the cross-linked construct) (pg. 11814 paragraph bridging column 1 & 2 lines 2-4 & 24-36; Table 1; Figure 4).
Regarding claim 3, Zhang teaches that duplex D3 (cross-linked construct) undergoes a conformational change (distortion) when higher voltage is applied enabling the cross-link to squeeze through the pore in which the double-stranded nucleic acid breaks in the vicinity of the interstrand cross-link as seen in Figure 4d (break in the vicinity of the interstrand cross-link occurs naturally due to distortion) (pg. 11814 paragraph bridging column 1 & 2 lines 2-4 & 24-36; Table 1; Figure 4).
Regarding claim 4, the specification of the instant application teaches that the nucleic acid can break by physically agitating the double stranded nucleic acid through an suitable for of physical agitation (pg. 31 of instant specification). The specification of the instant application also provides some examples of methods for physically agitating the double stranded nucleic acid, however these are not limiting definitions (pg. 31 of instant specification). Therefore, for the purposes of this rejection, physical agitation is given its broadest reasonable interpretation to encompass any physical shift in the double stranded nucleic acid.
Zhang teaches that duplex D3 (cross-linked construct) undergoes a conformational change (physical shift in the double stranded nucleic acid) when higher voltage is applied enabling the cross-link to squeeze through the pore in which the double-stranded nucleic acid breaks in the vicinity of the interstrand cross-link as seen in Figure 4d (break in the vicinity of the interstrand cross-link occurs by physical agitation) (pg. 11814 paragraph bridging column 1 & 2 lines 2-4 & 24-36; Table 1; Figure 4).
Regarding claim 11, Zhang teaches a method of preparing cross-linked DNA substrates through a reaction of the guanine residue with the aldehyde group of an abasic site on the opposing strand of the duplex DNA (interstrand cross-link is targeted to a particular base composition on the target double stranded nucleic acid) (pg. 11813 paragraph bridging column 1 & 2 lines 1-7).
Regarding claim 16, Zhang teaches method of preparing cross-linked DNA substrates in which the cross-link is positioned anywhere from 2-16 bases from the end of the duplex (cross-link is positioned within 100 bases of the terminus of the cross-linked construct) (Table 1).
Regarding claim 17, Zhang teaches examining the interstrand cross-links using a nanopore (nanopore sensor) for the detection and characterization (sequencing the cross-linked construct using a single molecule sequencing technique) (abstract lines 1-6; pg. 11817 paragraph bridging column 1 & 2 lines 1-19; Figure 2; Figure 4).
Claim(s) 7, 8, 12, & 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang et al.; ACS Nano, Vol. 9, pages 11812-11819, November 2015), and Crawford (WO 2015/150786 A1), as cited in the IDS dated 07/19/2022, as applied to claims 1-4, 11, 16, & 17 above, and further in view of Lai (Lai et al.; Analytical Chemistry, Vol. 80, pages 8790-8798, November 2008).
The teachings of Zhang and Crawford with respect to claim 1 are discussed above.
Regarding claims 7, 8, 12, & 15, Zhang and Crawford does not teach that the cross-linking agent comprises electromagnetic radiation (see claim 7) or comprises a chemical reagent (see claim 8). In addition, Zhang and Crawford does not teach that the cross-link is formed between nucleobases in the first and second strand of the target double stranded nucleic acid (see claim 12) or between nucleobase adducts in the first and second strands of the target double stranded nucleic acid (see claim 15).
Lai teaches a method for inducing interstrand cross links between the strands of a double stranded nucleic acid through contacting the double-stranded nucleic acids with psoralens (a chemical cross-linking reagent) and then exposing the double-stranded nucleic acid to UVA light (electromagnetic radiation) to form a cross-link between a thymine and another thymine in the opposing strand of the double strand nucleic acid (cross-link is formed between nucleobases in the first and second strand of the target double stranded nucleic acid) (abstract lines 1-15; pg.8790 paragraph bridging column 1 & 2 lines 1-9; pg. 8790 column 2 1st full paragraph lines 1-20; pg. 8796-8797 paragraph bridging pg. 8796 & pg.8797 lines 1-7). In addition, Lai teaches that the psoralens form monoadducts in which the monoadducts can form an interstrand cross-link between in the double stranded nucleic acid (cross-link is formed between nucleobase adducts in the first and second strands of the target double stranded nucleic acid) (abstract lines 1-15; pg. 8790 column 2 1st full paragraph lines 1-20; pg. 8796-8797 paragraph bridging pg. 8796 & pg.8797 lines 1-7). Finally, Lai teaches that this method provides a quantitative assessment understanding the properties of the DNA adducts and interstrand cross-links that form from psoralens and UVA exposure (pg. 8798 column 1 2nd full paragraph lines 6-11; pg. 8798 column 2 1st full paragraph lines 1-7).
Zhang, Crawford, and Lai are considered to be analogous to the claimed invention because they are all in the same field of analysis of target double stranded nucleic acids that are linked. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of preparing cross-linked DNA substrates in Zhang to incorporate the use of psoralens (chemical) and UVA light (electromagnetic radiation) as cross-linking agents for cross-linking nucleobases and nucleobase adducts between first and second strands of a double stranded nucleic acid as taught in Lai because Lai teaches that doing so would provide a method to quantitatively assess the properties of the interstrand cross-links formed.
Claim(s) 9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang et al.; ACS Nano, Vol. 9, pages 11812-11819, November 2015), and Crawford (WO 2015/150786 A1), as cited in the IDS dated 07/19/2022, as applied to claims 1-4, 11, 16, & 17 above, and further in view of Knox (Knox, Friedlos, Jarman, & Roberts; Biochemical Pharmacology, Vol. 37, pages 4661-4669, December 1988).
The teachings of Zhang and Crawford with respect to claim 1 are discussed above.
Regarding claim 9, Zhang and Crawford does not teach that the cross-linking agent comprises a nucleic acid cross-linking enzyme.
Knox teaches a monofunctional alkylating agent 5-(aziridine-1-yl)-2,4-dinitrobenzaminde (CB 1954) (a nucleic acid cross-linking enzyme) which forms DNA interstrand cross-links (abstract lines 1-4).
Zhang, Crawford, and Knox are considered to be analogous to the claimed invention because they are all in the same field of analysis of target double stranded nucleic acids that are linked. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of preparing cross-linked DNA substrates in Zhang to incorporate the use of 5-(aziridine-1-yl)-2,4-dinitrobenzaminde (CB 1954) as a nucleic acid cross-linking enzyme as taught in Knox as a means of simple substitution of one known element for another to obtain predictable results.
Claim(s) 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang et al.; ACS Nano, Vol. 9, pages 11812-11819, November 2015), and Crawford (WO 2015/150786 A1), as cited in the IDS dated 07/19/2022, as applied to claims 1-4, 11, 16, & 17 above, and further in view of Millard (Millard, Spencer, & Hopkins; Biochemistry, Vol. 37, pages 5211-5219, February 1998).
The teachings of Zhang and Crawford with respect to claim 1 are discussed above.
Regarding claim 10, Zhang and Crawford does not teach that the interstrand cross-link is formed at a random position on the target double stranded nucleic acid.
Millard teaches a method of interstrand cross-linking duplex DNA with nitrogen mustard family cross-linking reagent in which the cross-linking sites are distributed randomly throughout the sequence (abstract lines 1-5; pg. 5217 paragraph bridging column 1 & 2 lines 5-11). Millard also teaches that this method can provide an assessment on the susceptibility to conformational alterations in the cross-linked DNA and about cross-linking DNA in vivo with the antitumor cross-linking agents (pg.5212 paragraph bridging column 1 & 2 lines 19-34).
Zhang, Crawford, and Millard are considered to be analogous to the claimed invention because they are all in the same field of analysis of target double stranded nucleic acids that are linked. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of preparing cross-linked DNA substrates in Zhang to incorporate the use of a cross-linking that forms interstrand cross-links at random positions on the target double stranded nucleic acid as taught in Millard because Millard teaches that doing so would provide a method to determine conformational changes in the double stranded nucleic acid and assess the cross-linking agents in vivo.
Claim(s) 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang et al.; ACS Nano, Vol. 9, pages 11812-11819, November 2015), and Crawford (WO 2015/150786 A1), as cited in the IDS dated 07/19/2022, as applied to claims 1-4, 11, 16, & 17 above, and further in view of Ye (Ye et al.; Chemical Science, Vol. 4, pages 1319-1329, December 2012).
The teachings of Zhang and Crawford with respect to claim 1 are discussed above.
Regarding claim 13, Zhang and Crawford does not teach the interstrand cross-link is formed between sugar groups in the first and second strands of the target double stranded nucleic acid.
Ye teaches a method of forming interstrand cross-links between DNA strands through the amine group of the sugar group of the first strand of the target double stranded nucleic acid and then carboxylate group of the sugar group on the second strand of the target double stranded nucleic acid (interstrand cross-links is formed between sugar groups in the first and second strands of the target double stranded nucleic acid) (abstract lines 1-9; Figure 2). In addition, Ye teaches that this method of forming a interstrand cross-link between a double stranded nucleic acid molecules is site-specific, does not destabilize the rest of the DNA duplex, and only minimally perturbs the secondary structure (abstract lines 13-15).
Zhang, Crawford, and Ye are considered to be analogous to the claimed invention because they are all in the same field of analysis of target double stranded nucleic acids that are linked. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of preparing cross-linked DNA substrates in Zhang to incorporate the use of a cross-linking that forms interstrand cross-links between sugar groups in the first and second strands of the target double stranded nucleic acid as taught in Ye because Ye teaches that doing so would provide a method to interstrand cross-link a DNA duplex in a site specific manner with destabilizing the rest of the DNA duplex.
Claim(s) 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zhang (Zhang et al.; ACS Nano, Vol. 9, pages 11812-11819, November 2015), and Crawford (WO 2015/150786 A1), as cited in the IDS dated 07/19/2022, as applied to claims 1-4, 11, 16, & 17 above, and further in view of Lai (Lai et al.; Analytical Chemistry, Vol. 80, pages 8790-8798, November 2008) and Ye (Ye et al.; Chemical Science, Vol. 4, pages 1319-1329, December 2012).
The teachings of Zhang and Crawford with respect to claim 1 are discussed above.
Regarding amended claim 14, Zhang teaches a method of preparing cross-linked DNA substrates (pg. 11813 paragraph bridging column 1 & 2 lines 1-7; Scheme 1).
Zhang and Crawford does not teach that at least one multiple inter-strand cross-link is formed between nucleobases in the first and second strands of the target double stranded nucleic acid and at least one multiple inter-strand cross-link is formed between sugar groups in the first and second strands of the target double stranded nucleic acid.
Lai teaches a method for inducing interstrand cross links between the strands of a double stranded nucleic acid through contacting the double-stranded nucleic acids with psoralens and then exposing the double-stranded nucleic acid to UVA light to form a cross-link between a thymine and another thymine in the opposing strand of the double strand nucleic acid (cross-link is formed between nucleobases in the first and second strand of the target double stranded nucleic acid) (abstract lines 1-15; pg.8790 paragraph bridging column 1 & 2 lines 1-9; pg. 8790 column 2 1st full paragraph lines 1-20; pg. 8796-8797 paragraph bridging pg. 8796 & pg.8797 lines 1-7). In addition, Lai teaches that this method provides a quantitative assessment understanding the properties of the DNA adducts and interstrand cross-links that form from psoralens and UVA exposure (pg. 8798 column 1 2nd full paragraph lines 6-11; pg. 8798 column 2 1st full paragraph lines 1-7).
Ye teaches a method of forming interstrand cross-links between DNA strands through the amine group of the sugar group of the first strand of the target double stranded nucleic acid and then carboxylate group of the sugar group on the second strand of the target double stranded nucleic acid (interstrand cross-links is formed between sugar groups in the first and second strands of the target double stranded nucleic acid) (abstract lines 1-9; Figure 2). In addition, Ye teaches that this method of forming a interstrand cross-link between a double stranded nucleic acid molecules is site-specific, does not destabilize the rest of the DNA duplex, and only minimally perturbs the secondary structure (abstract lines 13-15).
Zhang, Crawford, Lai, and Ye are considered to be analogous to the claimed invention because they are all in the same field of analysis of target double stranded nucleic acids that are linked. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of preparing cross-linked DNA substrates in Zhang to cross-linking nucleobases between first and second strands of a double stranded nucleic acid as taught in Lai because Lai teaches that doing so would provide a method to quantitatively assess the properties of the interstrand cross-links formed and it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of preparing cross-linked DNA substrates in Zhang to incorporate the use of a cross-linking that forms interstrand cross-links between sugar groups in the first and second strands of the target double stranded nucleic acid as taught in Ye because Ye teaches that doing so would provide a method to interstrand cross-link a DNA duplex in a site specific manner with destabilizing the rest of the DNA duplex.
Response to Arguments
The response traverses the rejection. The response asserts that there is no reason for one of ordinary skill in the art to combine Zhang with Crawford when developing a method for sequencing double stranded polynucleotide sequence by cross-linking the two strands of the target polynucleotide and further, even if Zhang and Crawford were combined, one of ordinary skill would not have arrived at the claimed method with a reasonable expectation of success. Specifically, the response asserts that Zhang discloses that DNA fragments having inter-strand cross-links produce a current block when passing through a nanopore and that when a higher voltage was applied the duplex D3 was able to squeeze through the nanopore, however, the duplex D3 does not pass through the nanopore sequentially to enable sequencing of the target double stranded nucleic acid that has an inter-strand cross-link. Further, the response asserts that when a high voltage was applied both strands were able to squeeze through and not sequentially of the first strand and the second strand passed through the nanopore and that there is no disclosure in Zhang that such translocation would produce any sensible signal to discern sequence information, let alone producing orthogonal proof-reading information. Further, the response also asserts that Crawford discloses attaching a hairpin adapter to a double stranded polynucleotide and using a polynucleotide binding protein (enzyme) to unzip the double stranded polynucleotide such that the first and second strand are processed sequentially through the nanopore, however there is no disclosure in Crawford that the polynucleotide binding protein (enzyme) is able to process a cross-link through a nanopore and therefore one or ordinary skill in the art would not have had any reasonable expectation that the polynucleotide binding protein (enzyme) can process the inter-strand cross-link. These arguments have been thoroughly reviewed but were not found persuasive. First, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references as Zhang was not relied upon solely to teach producing orthogonal proof-reading information and Crawford is not relied upon solely to teach processing a cross-link through a nanopore. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Second, Zhang does teach sequential processing of the first and second strand of the cross-linked double stranded polynucleotide as Zhang teaches that when higher voltage is applied the D3 cross-link (cross-linked construct) conformational adjusts to enable the cross-link to squeeze through the pore in which the double-stranded nucleic acid breaks in the vicinity of the interstrand cross-link, as seen in Figure 4d-2, and then allows the cross-linked duplex D3 (cross-linked construct) to translocate from the cis to the trans side of the nanopore wherein the first strand, then the cross-link, and then the second strand is processed sequentially from one side of the nanopore to the other side of the nanopore and Zhang teaches that this produces specific signals as shown in Figure 4c. Further, there is a reasonable expectation of success as Zhang and Crawford are all in the same field of sequencing target double stranded nucleic acids through a nanopore in which Zhang teaches that a cross-linked target double stranded nucleic acid can be processed sequentially through a nanopore and Crawford teaches that that the movement of a linked target double stranded nucleic acid can be controlled with an enzyme. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of sequentially processing the first strand and the second strand in the cross-linked D3 construct in Zhang to incorporate the contacting the target double stranded nucleic acid with an enzyme as taught in Crawford to provide orthogonal proof-reading sequence information because Crawford teaches that doing so would provide a means to control the movement of the target double stranded nucleic acid through the nanopore.
The response also asserts that the deficiency of Zhang is discussed above and that Lai does not remedy the deficiencies of Zhang at least because Lai does not disclose using an enzyme that sequentially process the first and second strand of the cross-linked double stranded nucleic acid construct and therefore claims 7, 8, 12, and 15 are not obvious over Zhang in view of Lai. In addition, the response asserts that Knox does not remedy the deficiencies of Zhang at least because Knox does not disclose using an enzyme that sequentially process the first and second strand of the cross-linked double stranded nucleic acid construct and therefore claim 9 is not obvious over Zhang in view of Knox. In addition, the response asserts that Millard does not remedy the deficiencies of Zhang at least because Millard does not disclose using an enzyme that sequentially process the first and second strand of the cross-linked double stranded nucleic acid construct and therefore claim 10 is not obvious over Zhang in view of Millard. In addition, the response asserts that Ye does not remedy the deficiencies of Zhang at least because Ye does not disclose using an enzyme that sequentially process the first and second strand of the cross-linked double stranded nucleic acid construct and therefore claim 13 is not obvious over Zhang in view of Ye. These arguments have been thoroughly reviewed but were not found persuasive for the reasons set forth above.
For these reasons, and the reasons already made of record and modified to address the claims as currently amended, the rejections are maintained and applied to the newly amended claims.
Conclusion
Claims 1-4 & 7-17 are rejected.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BAILEY C BUCHANAN whose telephone number is (703)756-1315. The examiner can normally be reached Monday-Friday 8:00am-5:00pm ET.
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/BAILEY BUCHANAN/Examiner, Art Unit 1682
/JEHANNE S SITTON/Primary Examiner, Art Unit 1682